Variation in ORF3a Protein of SARS-CoV-2 Decreases The Severity of Host Cell Damage

INTRODUCTION: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) primarily targets the respiratory system. However, direct SARS-CoV-2 infection and viral protein expression have also been reported in other organs, which potentially contributes to multi-organ dysfunction and increased mortality in COVID-19. Since the original Wuhan-type genome was sequenced, several genetic variants of SARS-CoV-2 have emerged with differing pathophysiological properties such as the levels of transmissibility, disease severity, and mortality. We previously reported that the protein encoded by open reading frame 3a (ORF3a), a critical protein for SARS-CoV-2 replication and release, is found in the mitochondria of host cells and could increase oxidative stress and apoptotic signaling. ORF3a-Q57H is a highly recurrent variation and the most commonly found variant of ORF3a. Interestingly, the Q57H variant is associated with increased transmissibility, but lower mortality. AIM: To investigate the impact of the ORF3a-Q57H variant on host cell damage. METHODS: Plasmids carrying ORF3a-Q57H were generated by PCR-based site mutagenesis using Wuhan-type ORF3a (ORF3a-WT) as a template. Whole cell lysates were prepared from HEK293T cells and H9c2 cardiac myoblasts expressing ORF3a-WT or the mutant ORF3a-Q57H and used for biochemical assays. Live cell imaging for assessing subcellular localization of ORF3a/ORF3a-Q57H, mitochondrial reactive oxygen species (mROS), and caspase 3 activity in H9c2 cells was performed by confocal microscopy. RESULTS: In whole cell lysates, we found that ORF3a-Q57H exhibits significantly higher protein expression compared to ORF3a-WT. However, there is no significant difference in the ability of mitochondrial trafficking between ORF3a-WT and ORF3a-Q57H assessed by live cell imaging using GFP-tagged ORF3a/ORF3a-Q57H with mitochondria-targeted DsRed. Next, we investigated the effects of ORF3a expression on apoptotic and mitophagy signaling by quantifying the caspase 3 activity and LC3A/B ratio. We found that ORF3a-Q57H has significantly lower apoptosis and mitophagy signaling compared to WT despite its higher protein expression levels. ER-stress signaling was not activated in either ORF3a-WT or ORF3a-Q57H, as assessed by markers including Glucose-regulated protein 78/94 and C/EBP-homologous protein. Lastly, live cell imaging using the mitochondrial superoxide-sensitive dye Mitosox Red revealed that ORF3a-WT significantly increases mROS levels, but ORF3a-Q57H expression does not. CONCLUSION: SARS-CoV-2-ORF3a-Q57H causes lower oxidative stress and cell damage compared to the Wuhan-type variant, which could help explain the decreased mortality associated with the ORF3a-Q57H variant. These results provide novel insights on how genetic variations of SARS-CoV-2 influence the pathophysiology and clinical severity of COVID-19.